The induction of many diseases in mice by murine leukemia viruses (MuLVs), involves the participation of variant retroviruses termed polytropic MuLVs. These include the induction of proliferative, immunological and neurological disorders. Polytropic MuLVs are formed by recombination of exogenous ecotropic MuLVs with endogenous envelope sequences present in the genomes of inbred mouse strains resulting in viruses which utilize a distinct cell-surface receptor for infection. The infectious host range of ecotropic MuLVs is limited to mice;however the recombinant polytropic viruses generated after inoculation of mice with ecotropic MuLVs are capable of infecting a number of other species as well as mice. Thus, the generation of polytropic viruses results in a mixed retrovirus infection of viruses with different infectious properties. Our earlier studies strongly suggest that the interactions of ecotropic and polytropic MuLVs in the host play a role in facilitating oncogenesis. More recently we have investigated the interactions of retroviruses in mixed infections in vivo by co-inoculation of mice with mixtures of polytropic MuLV isolates and ecotropic MuLVs. Mice infected with defined mixtures of retroviruses exhibit dramatically altered pathology compared to infection with the individual viruses of the mixture. These included a highly significant delay in the induction of proliferative disease with one polytropic MuLV and a profound synergistic effect resulting in the abrupt development of a neurological disease with another polytropic isolate. In both instances the polytropic virus load in the co-inoculated mice was markedly enhanced while the level of the ecotropic MuLV was unchanged. Furthermore, the polytropic MuLV was nearly completely pseudotyped within ecotropic virions in co-inoculated mice. There are a number of possible mechanisms which could facilitate the profound in vivo amplification of the polytropic MuLVs including enhanced spread of the virus due to pseudotyping within ecotropic virions or possibly transactivation of the polytropic virus in co-infected cells. To examine these questions in a less complex system we have extended these studies to examine mixed retrovirus infections of in vitro cell lines. In 2011 we have extended earlier studies indicating that that mixed infections of in vitro cell lines with ecotropic and polytropic MuLVs resulted in amplification and pseudotyping characteristics remarkably similar to what we have observed in vivo. In all cases of mixed infections of cell lines with ecotropic MuLVs with polytropic MuLVs the polytropic MuLV genome was extensively pseudotyped within ecotropic virions and the infectious polytropic virus titer was profoundly elevated in co-infected cells. An amphotropic MuLV in mixed infections with a polytropic MuLV yielded similar results. In this case, the polytropic MuLV was pseudotyped within amphotropic virions and the infectious polytropic virus titer elevated. This result indicates that the amplification of the polytropic MuLV in mixed infections is not restricted to combinations of the polytropic virus with its ecotropic parent. The elevation of polytropic MuLV infectivity released from co-infected cells could have resulted from an increase in the level of polytropic genomes released. Alternatively, the observed increase could reflect a much higher specific infectivity of ecotropic or amphotropic virions compared to polytropic virions. We have found in polytropic MuLV mixtures with either ecotropic or amphotropic MuLVs, that much of the increase in polytropic virus titer can be attributed to an increase in the efficiency of packaging and release of the polytropic genome from co-infected cells. Studies utilizing clonal cell lines releasing different levels of polytropic viruses indicated that each of these lines could be induced to release similar high levels of polytropic virus upon co-infection of these cells with an ecotropic virus. These results further suggest that co-infection with an ecotropic virus facilitates the packaging and release of the polytropic genome.